Lipopolysaccharide-targeted peptide mimic vaccine against Q fever

ABSTRACT

A vaccine and method of vaccination for conferring immunity to Q fever is described. The vaccine comprises a polypeptide with a sequence of SLTWHKHELHRK (SEQ ID NO: 7) (m1E41920) or SPPWHKHELHRK (SEQ ID NO: 8) (m1E44), or at least 90% identity to m1E41920 or m1E44. Constructs are also provided for use in vaccination and treatment of Q fever. A method to identify and generate new vaccines to prevent diseases caused by intracellular Gram-negative bacteria is also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/338,730, filed Jul. 23, 2014, entitled “Lipopolysaccharide-TargetedPeptide Mimic Vaccine Against Q Fever,” which claims priority to U.S.Provisional Patent Application having Ser. No. 61/958,227, filed on Jul.23, 2013, each of which is hereby incorporated herein by reference inits entirety.

GRANT STATEMENT

This invention was made with Government support under Grant No. RO1AI083364 and Grant No. R21 AI075175 awarded by the National Institutesof Health. The Government has certain rights in the invention.

FIELD OF INVENTION

The present invention relates to a vaccine for preventing diseasesrelated to natural and/or deliberate exposure to an intracellularGram-negative bacterium, more specifically to a peptide mimic vaccineagainst Q fever.

SEQUENCE LISTING

A text file in compliance with ASCII and having a “.txt” extension hasbeen electronically submitted via EFS-Web. The text file named “SequenceListing” was created on Jun. 28, 2016, and is 12.8 KB. The text file isexpressly incorporated by reference herein in its entirety.

BACKGROUND

Coxiella burnetii is a Gram-negative bacterium that causes acute andchronic Query fever (Q fever) in humans. Q fever can be asymptomatic orit can cause flu-like symptoms, such as high fever, chills, sweating,fatigue, muscle pain, headache, loss of appetite, nausea and vomiting.These acute symptoms can last one to two weeks. In chronic cases,infected persons can develop liver and heart disease and in some cases Qfever can be fatal. In these chronic cases, a small percentage ofpatients develop hepatitis or liver disease and jaundice. Another raresymptom is endocarditis, an inflammation of the lining of the heartcavity. Animals such as cattle, sheep and goats can carry C. burnetii intissues involved in birth, such as the uterus, placenta, and birthfluids. Infected animals also release C. burnetii in milk and manure.Thus, people can acquire Q fever by inhaling infectious aerosols andcontaminated dusts generated by animals or through animal products.

C. burnetii is an obligate intracellular Gram-negative bacterium that isan understudied category B select agent and can be transmitted viaaerosol. The highly infectious nature of C. burnetii and its hardinessin adverse environmental conditions make this organism an importantzoonotic pathogen, and potentially useful in bioterrorism and biologicalwarfare. Since Q fever is a significant occupational hazard amongveterinarians, meat processing plant workers, sheep and diary workers,livestock farmers, and researchers at facilities housing sheep, C.burnetii has been used for developing biological weapons in the past,and recently chronic Q fever cases have been increasing worldwide, Qfever is becoming a significant public health concern. Because C.burnetii infection can develop into more severe chronic diseases,vaccination is the logical approach to prevent individuals at risk ofnatural and deliberate exposure. Although formalin-inactivated C.burnetii phase I (PI) whole-cell vaccine provides near-completeprotection in animal models as well as humans, it can induce severelocal or systemic adverse reactions when administered to individualswith prior immunity to the agent. A formalin-inactivated whole cellvaccine, Q-vax, has been developed and widely used in high-riskindividuals in Australia since 1989. Safe use of this vaccine requiresscreening of potential vaccines by skin test, serological tests, or invitro lymphocyte proliferation assay. The time consuming and costlyscreening procedures limit the use of phase I vaccine (PIV) for a massvaccination program. Currently, there is no licensed vaccine forpreventing Q fever in the United States. Creation of a safe andeffective new generation vaccine to prevent Q fever remains an importantpublic health goal.

Based on prior studies, C. burnetii undergoes a lipopolysaccharide (LPS)phase variation in which its virulent smooth LPS phase I (PI) convertsto an avirulent rough LPS phase II (PII) upon serial passage in eggs andtissue culture. It has been shown that PI vaccine (PIV) was moreprotective than PII vaccine (PIIV) in guinea pig and mouse models. Anearlier study has shown that PI-LPS is able to elicit antibody (Ab)responses to PI and PII antigens and to confer protection againstvirulent C. burnetii challenge in a mouse model. One recent study alsodemonstrated that PI-LPS induced a level of protection similar to PIV,but PII-LPS did not provide measurable protection. Since cultivation ofC. burnetii is difficult, hazardous, and requires the use of a BL3facility, it is very difficult to generate large quantities of purifiedbacteria for isolation of LPS, which consequently limits the use of C.burnetii LPS to produce vaccines. Thus, a LPS-based Q fever vaccine hasnot been successful.

BRIEF SUMMARY

In one embodiment of the invention, a novel LPS-based vaccine and methodof vaccination or conferring immunity against Q fever is described. Theinventive vaccine comprises a polypeptide with a sequence ofSLTWHKHELHRK (SEQ ID NO: 7) (m1E41920) or SPPWHKHELHRK (SEQ ID NO: 8)(m1E44), or at least 90% identity to m1E41920 or m1E44. According to oneembodiment of the invention, the inventive vaccine against Q fevercomprises a peptide m1E41920 conjugated to Keyhole limpet hemocyanin(KLH).

Other embodiments provide a method to identify and generate new vaccinesto prevent diseases caused by intracellular Gram-negative bacteria.Methods based on peptide-mimic technology for identifying and generatingnew vaccines to prevent diseases caused by LPS-based intracellularGram-negative bacteria are described. The method comprises the steps ofidentifying a protective monoclonal antibody specific to a LPS of aspecific intracellular Gram-negative bacteria that causes the specificLPS-based intracellular Gram-negative bacterial infection in thesubject; identifying one or more mimetic peptides by screening a PhageDisplay Library with the protective monoclonal antibody; and evaluatingthe one or more mimetic peptides to identify the one or more mimeticpeptides that confer an effective protective antigen against thespecific LPS-based intracellular Gram-negative bacterial infection inthe subject.

Embodiments of the present invention provide methods and constructs forinhibiting, vaccinating and treating a C. Brunetti infection. A PI-LPSspecific monoclonal antibody 1E4 that was developed. The PI-LPS specificmonoclonal antibody 1E4 is capable of inhibiting C. burnetii infectionin mice. In another embodiment a mutant mouse antibody (muscFv1E4) and amutant human antibody (huscFv1E4) were constructed and characterized.Both muscFv1E4 and huscFv1E4 were able to bind m1E41920 and live C.burnetii. Furthermore, muscFv1E4 and huscFv1E4 inhibits C. burnetiiinfection in mice and in mouse Bone Marrow-Derived Macrophages (BMDM) invitro. Interestingly, huscFv1E4 inhibits C. burnetii infection in humanmacrophages in vitro. In one embodiment, humanized 1E4 (huscFv1E4) canbe administered as a vaccine for preventing human Q fever.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Embodiments are described in detail below with reference to the attacheddrawing figures and pictures, wherein:

FIGS. 1(A) and 1(B) illustrate the generation of the monoclonal antibody(mAb) 1E4.

FIGS. 2(A) and 2(B) illustrate the characterization of (mAb) 1E4 byELISA and Western blotting.

FIGS. 3(A) to 3(C) evaluate the ability of 1E4 to inhibit C. burnetiiinfection in vivo.

FIGS. 4(A) to 4(E) evaluate the ability of 1E4 to confer protection inmice against C. burnetii aerosol infection.

FIGS. 5(A) to 5(C) represent the analysis of V_(H) and V_(L) gene usageand CDR sequence of 1E4.

FIGS. 6(A) and 6(B) compare the CDR amino acid sequence and structure ofmuscFv1E4 with huscFv1E4.

FIG. 7 illustrates the procedure for identifying the peptides viascreening.

FIGS. 8(A) and 8(B) illustrate the further identification of1E4-specific phage clones.

FIGS. 9(A) and 9(B) evaluate the binding abilities of the syntheticmimic peptide m1E41920 and m1E41920-KLH conjugate with 1E4 by ELISA andcompetitive ELISA.

FIGS. 10(A) to 10(C) are the analyses of m1E41920-KLH elicited antibodyresponse to PI antigen.

FIGS. 11(A) to 11(C) evaluate the ability of immune sera fromm1E41920-KLH-immunized mice to inhibit C. burnetii infection in BALB/cmice, both splenomegaly and with real-time-PCR.

FIGS. 12(A) to 12(D) evaluate the protective efficacy of m1E41920-KLHagainst C. burnetii infection in BALB/c mice.

FIGS. 13(A) and 13(B) are models of 1E4-m1E41920 complex established bythe molecular modeling and docking procedures.

FIGS. 14(A) to 14(D) characterize Fab1E4, muscFv1E4 and huscFv1E4.

FIGS. 15(A) to 15(E) evaluate the ability of 1E4, Fab1E4, muscFv1E4 andhuscFv1E4 to inhibit C. burnetii infection in vivo.

FIGS. 16(A) to 16(C) evaluate the ability of 1E4, Fab1E4, muscFv1E4 andhuscFv1E4 to inhibit C. burnetii infection in mouse BMDM.

FIGS. 17(A) to 17(C) evaluate the ability of huscFv1E4 to inhibit C.burnetii infection in human macrophages.

DETAILED DESCRIPTION

Coxiella burnetii is a Gram-negative bacterium that causes acute andchronic Q fever in humans. Creation of a safe and effective newgeneration vaccine to prevent Q fever remains an important public healthgoal. Previous studies suggested that Ab-mediated immunity to C.burnetii phase I (PI-LPS) is protective. To identify the potentialpeptides that can mimic the protective epitopes on PI-LPS, aPI-LPS-specific mAb 1E4 was generated, characterized, and used to screena phage display library. A mimetic peptide, m1E41920, inhibited C.burnetii infection in mice, suggesting that m1E41920 may specificallymimic the protective epitope of PI-LPS. Furthermore, 1E4 itself was ableto inhibit C. burnetii infection in mice. To further understand themechanisms of 1E4-mediated protection and to prove the feasibility ofusing 1E4 to prevent human Q fever, the Fab fragment of 1E4 (Fab1E4),recombinant murine single chain variable fragments (muscFv1E4) wasexamined and humanized single chain variable fragments (huscFv1E4) andfound to retain the ability of 1E4 to inhibit C. burnetii infection. Theresults indicated that Fab1E4, muscFv1E4 and huscFv1E4 were able toinhibit C. burnetii infection in mice. Interestingly, treatment of C.burnetii with huscFv1E4 can significantly reduce C. burnetii infectivityin human macrophages.

A PI-LPS Specific Monoclonal Antibody 1E4

The C. burnetii stain used in these studies was C. burnetii Nine Milephase I (PI) clone 7 (RSA493), which was propagated in L929 cells andpurified by sucrose density centrifugation. Purified PI organisms wereinactivated by 1% formaldehyde solution and used as whole cell antigenfor ELISA. The protein concentration of inactivated PI whole cellantigen was measured by a Micro BCA™ Protein Assay Kit (Pierce,Rockford, Ill.).

Specific pathogen-free (SPF) 8 weeks old female BALB/c and SCID(CBySmn.CB17-Prkdc^(scid)/J) mice were purchased from the JacksonLaboratory (Bar Harbor, Me.). All mice were housed in sterilemicroisolator cages under SPF conditions in laboratory animal facilityaccording to the Guide for the Care and Use of Laboratory Animals at theUniversity of Missouri. The experimental protocols described in thisreport were approved by the Institutional Biosafety Committee and theAnimal Care and Use Committee of MU. All C. burnetii infectionexperiments were conducted in Animal Biohazard Safety Level 3 (ABL3)facilities at the MU Laboratory of Infectious Disease Research (LIDR).

One embodiment of the invention discloses a novel LPS-based vaccineagainst Q fever. The inventive LPS-based vaccine is safe, effective, andwithout inducing severe local or systemic adverse reactions whenadministered to a subject. A subject can be any animal, including mice,guinea pigs, chimpanzees, humans, and the like. To develop the LPS-basedvaccine, the novel PI-LPS specific monoclonal antibody (mAb) 1E4 wasidentified.

Turning to FIGS. 1(A) and 1(B), to generate the monoclonal antibody(mAb) 1E4 against PI-LPS, 6-wk-old BALB/c mice were immunized with 10 μgformalin-inactivated C. burnetii NMI Ag four times at 3-wk intervals andused to isolate splenocytes. To develop monoclonal antibodies, thehybridomas were obtained from the fusion of splenocytes fromPI-immunized BALB/c mice and SP2/0 myeloma cells, and then hybridomasupernatants were screened by ELISA for their ability to react with C.burnetii PI and PII antigens. FIG. 1(B) illustrates that ELISA-positivehybridoma cell populations were subcloned and cultured, whereas a totalof 14 positive hybridomas were cloned. The positive hybridomas werecloned by limiting dilution and isotyped by ELISA. Cloned hybridomaswere also analyzed by immunoblotting with proteinase K-treated anduntreated PI and PII antigens.

One hybridoma, 1E4, which recognizes PI-LPS, was cultured in HybridomaSerum Free Medium (Invitrogen) and purified from the supernatants byusing HiTrap protein G HP columns (GE Healthcare) according to theprotocol from manufacture. Purified 1E4 was desalted and concentrated byusing an Amicon Ultra-15 centrifugal filter device with a 30 kDamolecular-weight cutoff (Millipore). The purity of the purified 1E4 wasanalyzed by Coomassie blue-staining of SDS-PAGE gel and the proteinconcentration of 1E4 was measured by the Micro BCA™ Protein Assay Kit.

Turning to FIGS. 2(A) and 2(B), illustrated is the characterization of(mAb) 1E4 by ELISA and Western blotting. As shown in FIG. 2(A), isotypeof 1E4 by ELISA indicated that in PI antigen coated plate, 1E4 reactedwith anti-IgG and -IgG2a secondary antibodies but did not react withanti-IgM, -IgG1, -IgG2b and -IgG3 secondary antibodies. This resultsuggests that the isotype of 1E4 is IgG2a. As shown in FIG. 2(B),Western blotting analysis demonstrated that 1E4 specifically reactedwith a 14 kDa band in both PI and proteinase K digested PI antigens butdid not react with PII, suggesting that 1E4 specifically recognizesepitopes on PI-LPS.

C. burnetii infection in mice does not cause death and clear clinicalsigns. However, infection can induce significant splenomegaly.Splenomegaly has been used as an indicator to monitor severity of C.burnetii infection in mice. In addition, because C. burnetii isdifficult to grow on a plate and does not form clear plaques in cellculture, traditional methods cannot be used to measure the C. burnetiiburden in animal tissues. Recently, a quantitative real-time PCRprocedure has been developed and used to accurately measure the numberof C. burnetii in the spleen. It was shown that splenomegaly iscorrelated with infection dose and also correlated with bacterialloading in the spleen, as measured by real-time PCR. This suggests thatsplenomegaly can be a useful indicator to monitor severity of infectionand may be useful to evaluate the protective efficacy of vaccine. Inthis study, we used splenomegaly and bacterial burden and pathologicalchanges in the spleen to evaluate the ability of 1E4 in inhibiting C.burnetii infection and the protective efficacy of mimic peptidevaccine-induced protection against C. burnetii challenge in BALB/c mice.The results provided additional evidence to support that mousesplenomegaly sublethal challenge model can be used to measure theability of Ab-mediated protection and the protective efficacy of vaccinecandidates against C. burnetii infection. In addition, our resultssuggest that pathological change in the spleen can be used as anadditional indicator to evaluate the ability of Ab-mediated protectionand the protective efficacy of vaccines against C. burnetii infection.

In one embodiment, with reference to FIGS. 3(A) and (B) it wasdemonstrated that a PI-LPS specific monoclonal antibody (mAb) 1E4 wasable to inhibit C. burnetii infection in vivo; passive transfer of 1E4was able to confer significant protection against Q fever in mice. FIGS.3(A) and 3(B) show the ability of 1E4 to inhibit C. burnetii infectionin vivo. The inhibition of C. burnetii was performed by incubation of1×10⁷ virulent C. burnetii NMI with 1, 10, 100 and 300 μg of purified1E4, or mouse IgG2a isotype control at 4° C. overnight. Splenomegaly andbacterial burden in the spleen were measured at 14 days post infectionand used as indicators to evaluate the ability of 1E4 to inhibit C.burnetii infection in BALB/c mice. Compared to mice infected with PBS ormouse IgG2a isotype control-treated C. burnetii, 1E4 was able to inhibitC. burnetii infection in a dose-dependent manner. The splenomegalyanalysis is included in the FIG. 3(A) plot, while the real-time-PCRanalysis is in FIG. 3(B), and both analyses demonstrate that 1E4 is aprotective mAb. For both FIGS. 3(A) and 3(B), *p<0.05 and **p<0.01. ThemAb 1E4 was able to inhibit C. burnetii infection in vivo in adose-dependent manner, suggesting that 1E4 may directly neutralize orbecome bactericidal toward C. burnetii to block C. burnetii infection.FIG. 3(C) shows pathological changes in the spleen of mice infected withIgG2a isotype control, 10, 100, or 300 μg 1E4-treated C. burnetii.Robust extramedullary hematopoiesis and increased macrophages with moreaggregates occurred in mice infected with PBS, IgG2a isotype control, orlower doses (1 or 10 μg) of 1E4-treated C. burnetii, whereas there weredecreased extramedullary hematopoiesis and few to no aggregates ofmacrophages in mice infected with 100 or 300 μg 1E4-treated C. burnetii.In addition, multifocal moderate to large accumulations of macrophages(arrow) were present throughout red pulp of spleens of mice infectedwith PBS, IgG2a isotype control, or lower doses (1 or 10 μg) of1E4-treated C. burnetii. In contrast, multifocal accumulations ofmacrophages (arrow) were fewer and smaller in red pulp of spleens ofmice infected with 100 μg of 1E4-treated C. burnetii. In the spleen ofmice infected with 300 μg 1E4-treated C. burnetii, there were only twosmall accumulations of macrophages present in red pulp of spleen of oneof four mice. Thus, pathological changes in the spleen correlated tosplenomegaly and bacterial burden in the spleen. These resultsdemonstrate that 1E4 was able to inhibit C. burnetii infection in vivoin a dose-dependent manner, suggesting that 1E4 may directly neutralizeor become bactericidal toward C. burnetii to block C. burnetiiinfection. FIG. 3(C) shows pathological changes in the spleen at 14 dayspostchallenge. The data presented in each group are the average with SDof four mice. Original magnification ×410.

It was shown that both passive transfer of immune sera or Abs andpremixed immune sera or Abs with C. burnetii were able to transfersignificant protection to naive recipient mice. Although the mechanismsof Ab-mediated protection against C. burnetii infection remain unclear,the Ab binding with bacteria is necessary for direct bactericide,neutralization, or opsonization. To reduce the influence ofunpredictable factors in in vivo variables, such as Ig biodistributionand catabolism on the Ab-pathogen complex formation, immune sera, mAbwere premixed with C. burnetii for evaluating its ability to inhibit C.burnetii infection in this study. In addition, premixing immune sera ormAb with C. burnetii may be able to provide quantitative measurement oftheir ability to inhibit C. burnetii infection.

As shown in FIG. 4A, to determine whether passive transfer of Abs canprovide protection against C. burnetii natural infection, we examined ifpassive transfer of 1E4 would provide significant protection against C.burnetii aerosol challenge in SCID mice, compared to mice receiving PBSor mouse IgG2a isotype control, splenomegaly was significantly reducedin mice receiving 1E4 (p<0.001). In FIG. 4A, as compared to PBS or mouseIgG2a isotype control recipient mice, significantly lower C. burnetiigenome copies were detected in spleens and lungs from 1E4 recipient mice(p<0.001, FIGS. 4(B) & (C)). In addition, differences in interstitialinflammation as measured by the concentration of macrophages andneutrophils in interalveolar septum and alveolar spaces were observed inthe lungs between PBS or IgG2a and 1E4 receiving mice (FIG. 4(D)).Moderate to large accumulations of macrophages and neutrophils wereobserved in the lungs from PBS or IgG2a receiving mice, which affectedmore than 10% of lung parenchyma. In contrast, few small scatteredaccumulations of neutrophils and macrophages of mild to moderateaccumulations of macrophages and neutrophils were present in the lungsfrom mice receiving 1E4, which affected less than 10% of lungparenchyma. The severity of inflammation in the lung was significantlyreduced in 1E4 receiving mice as compared with PBS or IgG2a receivingmice (p<0.01, FIG. 4E). These observations indicated that passivetransfer of 1E4 provided protection against aerosolized C. burnetiichallenge-induced inflammatory response in the lung.

These results also demonstrated that passive transfer of PI-LPS specificmAb 1E4 was able to confer significant protection against aerosolized C.burnetii in naïve recipient mice, suggesting that the C. burnetiiaerosol infection mouse model can be used to evaluate the efficacy ofAb-mediated protection against C. burnetii natural infection. Passiveimmunization of 1E4 was performed by i.p. injection of 300 μg ofpurified 1E4 into each SCID mouse. In addition, SCID mice receiving 300μg of control IgG2a or PBS were used as negative controls. All mice wereexposed to virulent PI using a Liquid Sparging Areosolizer. Thisapparatus ensures a uniform dose of bacteria to the lower airways thougha nose-only aerosol exposure. A total of 1×10⁹ bacteria resuspended in 5ml of PBS was aerosolized and used to challenge SCID mice. The totalexposure time was approximately 30 minutes. Samples collected frombubbling the aerosol through PBS showed that of the 1×10⁹ startingconcentration, each mouse actually received approximately 1×10⁷ C.burnetii. Mice were sacrificed at 14 days post infection. Mouse body andspleen weights were measured, and a portion of lung and spleen from eachmouse was collected for real-time-PCR analysis. The ability of 1E4 toconfer protection against C. burnetii aerosol infection was evaluated bycomparing splenomegaly, bacterial burden and histopathological changesin the lung and spleen at 14 days post infection with controls.

TABLE 1 Primer Sequence FVLK 5′-gac att gag ctc acc cag tct cca-3′ RCLK5′-ggc tcg agg aag atg gat aca gtt ggt  gca-3′ FVH15′-ag gtg cag ctc gag gag tca gga cc-3′ FVH25′-gag gtc cag ctc gag cag tct gga cc-3′ FVH35′-cag gtc caa ctc gag cag cct ggg gc-3′ FVH45′-gag gtt cag ctc gag cag tct ggg gc-3′ FVH55′-gag gtg aag ctc gag gaa tct gga gg-3′ FVH65′-gag gta aag ctc gag gag tct gga gg-3′ FVH75′-gaa tgt cag ctc gag gag tct ggg gg-3′ FVH85′-gag gtt cag ctc gag cag tct gga gc-3′ RCHg2a5′-gtt ctg act agt ggg cac tct ggg ctc-3′

The 1E4 has Unique V_(H) and V_(L) Gene Usage and CDR Sequence

The 1E4 V_(H) gene was only amplified by using FVH7-RCHg2a primer pairs(data not shown). As shown in FIG. 5(A), the IMGT/V-QUEST analysis of1E4 nucleotide sequences {GenBank accession numbers: JX139949 (V_(H)gene) and JX139950 (V_(L) gene)} and indicate that the 1E4 V_(H) gene isencoded by a rarely reported murine germline family IGHV13-2. BLASTsearch of the 1E4 V_(H) gene nucleotide and amino acid sequences in theGenBank non-redundant sequence databases found that only one reportedbacterial carbohydrate antigen specific antibody, the anti-Neisseriameningitidis group B polysaccharide mAb SEAM12 (GenBank: DQ113493.1),was derived from IGHV13-2. FIG. 5(B) shows the alignment analysis ofrespective amino acid sequences of 1E4, IGHV13-2 and SEAM12 V_(H) gene.Compared to the germline gene IGHV13-2, several mutations wereidentified in HCDR1 and HCDR2 regions of 1E4 and SEAM12 V_(H) genes.Interestingly, the HCDR3 region differed in length, pI andhydrophobicity between 1E4 and SEAM12 (FIGS. 5(B) and (C)). The HCDR3 of1E4 has 15 amino acids but there are only 10 amino acids found in theHCDR3 of SEAM12. The HCDR3 of 1E4 has a much lower pI (4.4) than theHCDR3 of SEAM12 (pI 10.75). 1E4 contains three highly hydrophobic valineamino acids and the average normalized Kyte-Doolittle hydrophobicity ofHCDR3 loop is greater than 1.12. In contrast, the SEAM12 HCDR3 containsthree highly charged arginine amino acids and the average hydrophobicityis just −1.29. FIG. 5: Panel A, analysis of 1E4 V_(H) and V_(L) geneusage by IMGT/V-QUEST. a: identity to germline genes. Panel B,comparison of amino acid sequence of 1E4 V_(H) gene with theanti-Neisseria meningitidis group B polysaccharide mAb SEAM12 and murinegermline family IGHV13-2 by ClustalW program. A star (*) indicates thesame amino acid residue identity in a position, while a dot (:)indicates a different residue, and a dash (-) indicates inserted spacesplaced in the sequence to provide maximum identity. Panel C, comparisonof the HCDR3 conjunction between 1E4 and SEAM12. N: nucleotide additionmutation; P: P deletion mutation.

Characterization of a Mutant Mouse Antibody (muscFv1E4) and a MutantHuman Antibody (huscFv1E4)

The Complementarity Determining Regions (CDRs) in the variable regionsof Abs play a critical role in their specificity and affinity by meansof shape and charge complementarities. To define the role of CDRs in theFab fragment of 1E4, a mutant mouse antibody (muscFv1E4) and a mutanthuman antibody (huscFv1E4) were designed and constructed by humanizedCDR grafting (FIG. 6A). The structure models of the muscFv1E4 andhuscFv1E4 were superimposed to determine conformational changes in theloop regions. As depicted in FIG. 5B, the muscFv1E4 CDR colored in blackshowed nearly identical loop structures compared to the huscFv1E4 CDRscolored in gray. The root mean square (RMS) which can be used to measurethe three dimensional structural similarity was calculated by SwissPdbViewer. Higher RMS values usually mean greater conformationaldissimilarity and 1.5 A° was defined as the Ab similarity cut-offvalues. The RMS of the CDR loops in muscFv1E4 and huscFv1E4 structuralalignment model ranged from 0.2 to 1.0 A° indicating a significantlysimilar conformation. However, as shown in FIG. 6B, an apparentdifference between muscFv1E4 and huscFv1E4 was observed in the heavychanges FR2 (HFR2) loop. SwissPdb Viewer calculation showedconformational changes at PPGKR residues of HFR2, resulting in a veryhigh average RMS value of 2.88 A°. These results indicate that exceptfor CDR domains, huscFv1E4 has less than 70% sequence identity to itsparent mAb 1E4 as well as a significant conformational change in theHFR2 region, suggesting that the muscFv1E4 derived mutationconstruction, huscFv1E4, may be useful for identifying the role of CDRsin the Fab fragment of 1E4. To purify the 1E4 and Fab fragment of 1E4,hybridoma 1E4 was cultured in Hybridoma Serum Free Medium (Invitrogen,Grand Island, N.Y.) and 1E4 was purified from the supernatants usingprotein G columns. The Fab fragment of 1E4 was generated by papaindigestion of 1E4 with an ImmunoPure Fab Preparation Kit (Thermo FisherScientific, Rockford, Ill.) and separated from undigested 1E4 and theconstant domain fragment (Fc) by using Protein A column. The flowthrough fraction was collected as the purified Fab fragment of 1E4. Thepurity of purified 1E4 and the Fab fragment were analyzed by SDS-PAGEand Western blotting. The concentrations of 1E4 and the Fab fragmentwere measured by Bradford assay (Bio-Rad, Hercules, Calif.).

To generate muscFv1E4 of 1E4, total RNA was extracted from ˜10⁵monoclonal 1E4 hybridoma cells using QIAgen RNeasy Mini Kit (Qiagen,Valencia, Calif., USA) and the cDNA was obtained using QuantiTectRevTranscription Kit (Qiagen) according to the manufacturer's instructions.The variable heavy chain (VH) and variable light chain (VL) genes wereamplified separately by PCR. The PCR products of VL and VH genes weregel purified using QIAquick gel extraction kit (Qiagen) and separatelycloned into pCR2.1-TOPO vector (Invitrogen). Randomly selected cloneswere sequenced using the M13 primers at the MU's DNA core facility. Thesequences of VL and VH genes were compared with 1E4 nucleotide sequencesin the Genbank (VL gene: JX139950 and VH gene: JX139949), respectively.The correct 1E4 VH and VL genes were designated as pTVHE4 and pTVLE4,respectively, and used as PCR templates to amplify full-length 1E4 scFvgene. Primers were designed to introduce Nde I restriction sites and thepelB leader sequence, respectively. One primer was designed to introducethe Hind III restriction sites. Primers were designed to carryoverlapping sequences encoding the link peptide (Gly₄Ser)₃. Theamplified full-length scFv gene was subcloned into the Nde I/Hind IIIsite of pET23a via overlap extension (SOE) methods (EMD Millipore,Darmstadt, Germany). The correct clone was selected based on DNAsequencing and used as the recombinant plasmid pETmuscFv1E4, in whichthe C-terminal domain of scFv was fused with a His-tag for improvedpurification and immune identification.

The construction of a humanized 1E4 scFv (huscFv1E4) by humancomplementarity determining region (CDR)-grafting was established asfollows: the VH and VK sequences of 1E4 (GenBank: JX139949 and GenBank:JX139950) were compared with the human VH and VK gene families usingNCBI IgBLAST tools. The human V genes frameworks IGHV3-30-3*01 (71%identical to 1E4 VH framework) and IGKV6D-41*01 (66% identical to 1E4 VLframework) were chosen to accept the 1E4 CDRs based on their highestamino acid sequence identity. Almost all of the framework amino acidresidues that are different between 1E4 and human sequences were changedto human sequences. Four murine residues, including VH2, VH48, VL2, andVL4, which belong to the “vernier zone” were retained at their positionsas “back mutation”, because these residues may strongly affect thestructure of CDRs and the antibody affinity. The designed Nde I/Hind IIIflanked humanized 1E4 scFv DNA sequence (the N terminus pelB leadersequence upstream from the humanized 1E4 VH chain—the (Gly₄Ser)₃ peptidelinker—the humanized 1E4 VL chain) was synthesized by GenScriptCorporation (Scotch Plaines, N.J., USA) and cloned into pUC57 vector,resulting in pUChuscFv1E4. The Nde 1/Hind III fragment of pUChuscFv1E4was then cloned into pET23a to generate the expression plasmidpEThuscFv1E4.

The automatic modeling of variable regions of muscFv1E4 and huscFv1E4were established by canonical structure method. The deduced VH and VLamino acid sequences of muscFv1E4 and huscFv1E4 were submitted to thePrediction of ImmunoGlobulin Structure (PIGS), respectively. Theresulting models were aligned and displayed by PyMOL (Delano Scientific,San Carlos, Calif.). In addition, the muscFv1E4 and huscFv1E4 modelswere superimposed by DeepView v4.1 to calculate the root mean square(RMS) for comparing their three dimensional structural similarity.

Identification of PI-LPS Mimetic Peptides

In one embodiment, peptide-mimic technology was employed for identifyingpeptide mimics on PI-LPS by screening a Phage Display Library with mAb1E4. Refer to FIG. 7, which illustrates the procedure for identifyingthe peptides via screening. To identify the peptides that can mimic theprotective epitopes of PI-LPS, the Ph.D.-12 Phage Display PeptideLibrary (New England Biolabs Inc., Beverly, Mass.) was screened with 1E4in a solid-phase support system. Three subsequent biopanning rounds werecarried out according to the manufacturer's instructions. Briefly, 100,10, and 1 μg/ml of 1E4 were used in the first, second and third roundpanning, respectively. Amplified phage clones were purified byprecipitating the phage particles with PEG/NaCl (20% polyethylene glycol8000 and 2.5 M NaCl). The selected phage DNAs were extracted by usingiodide buffer (10 mM Tris-HCl [pH 8], 1 mM EDTA, 4 M NaI) according tothe manufacturer's instructions. Sequence analysis of the peptideinserts was performed by automated dideoxyoligonucleotide sequencingusing the −96 M13 primer (New England Biolabs Inc.) at the MU's DNA corefacility. The inserted peptide sequences were deduced from the DNAsequences. A total of 20 phage plaques were randomly picked, amplifiedand sequenced.

The 20 phage plaques were further analyzed to identify the 1E4-specificphage clones: m1E41920 and m1E44 with the sequence, SLTWHKHELHRK (SEQ IDNO: 7) and SPPWHKHELHRK (SEQ ID NO: 8), respectively. Turning to FIGS.8(A) and 8(B), illustrated is the further identification of 1E4-specificphage clones. As shown in amino acid sequence alignment analysis (FIG.8(A)), m1E41920 and m1E44 have the closest alignment. A total of 16mimetic peptides (with unique sequences) that could bind to 1E4 wereidentified after three rounds of biopanning against 1E4. As shown inFIG. 8(B), each of the sequenced unique phage clones was analyzed byELISA to determine their ability to bind to 1E4. The results indicatethat all identified 1E4-specific phage clones were able to bind to 1E4.To determine whether identified 1E4-specific phage clones canspecifically mimic the protective epitopes of PI Ag, the selected phageclones were further tested for their binding ability with 1E4 to competeagainst binding with PI Ag by a competitive inhibition ELISA.Approximately 10¹⁰ of purified phage particles in 0.05 Mcarbonate/bicarbonate coating buffer (pH 9.6) were coated in a 96-wellmicrotiter plate at 4° C. overnight. One hundred microliters of 5 μg/ml1E4 with or without 2 μg/ml of PI antigen was added into the wells ofthe phages coated plate. The binding abilities of 1E4 with or without PIantigen were detected with horseradish peroxidase-conjugated anti-mouseIgG. The inhibition index (A490 without PI-A490 with PI/A490 without PI)was used to evaluate the ability of PI antigen to inhibit 1E4 bindingwith selected phage particles. FIG. 8(B) shows the ability of PI Ag toinhibit 1E4 binding with selected phage clones as measured by inhibitionindex (A490 without PI-A490, with PI/A490, without PI). The phageclones, m1E44 and m1E41920, showed the highest inhibition index,suggesting these two phage clones may contain mimic epitopes that bindto the same 1E4 binding site as PI-LPS.

The mimotope, m1E41920 has been chemically synthesized and conjugated toKeyhole limpet hemocyanin (KLH) for further examining itsimmunogenicity. Refer to FIGS. 9(A) and 9(B), which evaluate the bindingabilities of the synthetic mimic peptide m1E41920 and m1E41920-KLHconjugate with 1E4 by ELISA and competitive ELISA. The m1E41920,m1E41920-KLH, control peptide, control peptide-KLH, or KLH was coated ina 96-well microtiter plate at 4° C. overnight. The mAb 1E4 was addedinto the synthetic peptide-coated plate. The binding ability of 1E4 withsynthetic peptides was detected with HRP-conjugated anti-mouse IgG. Inaddition, the synthetic m1E41920 was further tested for its bindingability with 1E4 to compete against binding with PI Ag by a competitiveinhibition ELISA. The mAb 1E4 was mixed with different concentrations ofm1E41920 or control peptide and added into a 96-well microtiter platethat was coated with PI Ag. The ability of m1E41920 to inhibit 1E4binding with PI Ag was detected with HRP-conjugated anti-mouse IgG.ELISA analysis (FIG. 9(A)) indicated that both synthetic peptidem1E41920 and m1E41920-KLH conjugate specifically bound to 1E4,suggesting that m1E41920 and m1E41920-KLH conjugate retain their bindingability to 1E4. Competitive inhibition ELISA analysis (FIG. 9(B))demonstrated that m1E41920 was able to inhibit the binding of 1E4 to PIantigen in a dose dependent manner. These results suggest that syntheticm1E41920 peptide can mimic the C. burnetii specific epitope on PIantigen.

Another embodiment further provides the analyses of m1E41920-KLHelicited antibody response to PI antigen. Turning now to FIGS. 10(A) to10(C), ELISA analysis indicates that m1E41920-KLH was able to elicitspecific IgG response against PI antigen. Western blotting showed thatimmune sera from m1E41920-KLH-immunized mice reacted with a 14 kDaproteinase K resistant band, which is identical to 1E4 recognizedantigen. Competitive inhibition ELISA result demonstrated that m1E41920was able to inhibit the binding of immune sera fromm1E41920-KLH-immunized mice to PI antigen in a dose dependent manner,while the peptide control did not inhibit immune sera fromm1E41920-KLH-immunized mice bind to PI antigen. These results furtherprove that synthetic m1E41920 peptide mimics epitopes on PI-LPS.

In another embodiment, it has also been determined that m1E41920-KLH caninduce a specific Ab response against PI antigen. Turning to FIGS. 11(A)and 11(B), evaluating the ability of immune sera fromm1E41920-KLH-immunized mice to inhibit C. burnetii infection in BALB/cmice, both splenomegaly and with real-time-PCR, is shown. Compared tomice infected with normal mouse sera-treated C. burnetii, splenomegalyand bacterial burden in the spleen were significantly reduced in miceinfected with immune sera from PIV-vaccinated mice, 1E4 or immune serafrom m1E41920-KLH-immunized mice-treated C. burnetii. These resultsdemonstrate that immune sera from m1E41920-KLH-immunized mice were ableto inhibit C. burnetii infection, suggesting that m1E41920 specificallymimics the protective epitope on PI-LPS. For FIGS. 11(A) and 11(B),*p<0.05 and **p<0.01. As shown in FIG. 11(C), histopathologicaldifferences were observed in the spleen between mice infected withnormal mouse sera-treated C. burnetii and mice infected with immunesera-treated C. burnetii. There were more and larger multifocalaccumulations of macrophages (arrow) in red pulp of spleens of miceinfected with normal mouse sera-treated C. burnetii than mice infectedwith immune sera from PIV-vaccinated mice, 1E4, or immune sera fromm1E41920-KLH-immunized mice-treated C. burnetii. This indicates thatimmune sera from PIV-vaccinated mice, 1E4, or immune sera fromm1E41920-KLH-immunized mice provided similar levels of protectionagainst C. burnetii challenge-induced inflammatory responses. Theseresults indicate that immune sera from m1E41920-KLH-immunized mice wasable to inhibit C. burnetii infection in vivo, whereas immune sera fromPIV-vaccinated mice or 300 μg 1E4 had a stronger ability than immunesera from m1E41920-KLH-immunized mice to inhibit C. burnetii infection.These results suggest that m1E41920 specifically may mimic theprotective epitope of PI-LPS. For FIG. 10(C), pathological changes inthe spleen were at 14 days post-challenge. The data presented in eachgroup are the average with SD of four mice. Original magnification ×400.

One embodiment further teaches m1E41920-KLH is able to confer protectionagainst C. burnetii infection, thus a protective antigen against Qfever. Turning to FIGS. 12(A) to 12(C), evaluation of the protectiveefficacy of m1E41920-KLH against C. burnetii infection in BALB/c mice isshown. Compared to KLH-immunized control mice (FIG. 12(A)), splenomegalyand the bacterial burden in the spleen were significantly reduced inm1E41920-KLH-vaccinated mice (FIGS. 12(B) and 12(C)). This resultsuggests that m1E41920-KLH is a protective antigen and a candidate fordeveloping a safe and effective peptide mimic vaccine to prevent human Qfever. For FIGS. 12(A) to 12(C), *p<0.05, **p<0.01 and ***p<0.001. Inaddition, multifocal accumulations of large macrophages (arrow) werepresent in red pulp of spleens of KLH-immunized mice, but not in PIV- orm1E41920-KLH-immunized mice (FIG. 12(D)), which indicates that PIV- orm1E41920-KLH-immunized mice provided similar levels of protectionagainst C. burnetii challenge-induced inflammatory responses. Theseresults suggest that m1E41920-KLH was able to confer significantprotection against C. burnetii challenge, but the levels of protectionwere lower than PIV. For FIG. 12(D), pathological changes in the spleenwere at 14 days post-challenge. The data presented in each group are theaverage with SD of four mice. Original magnification ×400.

Another embodiment further provides a general method for identifying andgenerating new vaccines to prevent diseases caused by LPS-basedintracellular Gram-negative bacteria besides C. burnetii. The methodcomprises the steps of identifying a protective monoclonal antibodyspecific to a LPS of a specific intracellular Gram-negative bacteriathat causes the specific LPS-based intracellular Gram-negative bacterialinfection in the subject; identifying one or more mimetic peptides byscreening a Phage Display Library with the protective monoclonalantibody; and evaluating the one or more mimetic peptides to identifythe one or more mimetic peptides that confer an effective protectiveantigen against the specific LPS-based intracellular Gram-negativebacterial infection in the subject. An effective protective antigenagainst the specific LPS-based intracellular Gram-negative bacterialinfection in the subject is a protective antigen that prevents infectionin a subject by the Gram-negative bacterium.

In one embodiment, the Phage Display Library is a Ph.D.-12 Phage DisplayPeptide Library. One or more mimetic peptides from the screening of thePhage Display Peptide Library comprise the following peptide sequences:SWFHPQRRHSHQ (SEQ ID NO: 9), SWMPHPRWSPQH (SEQ ID NO: 10), MHRAPSTHKLLP(SEQ ID NO: 11), ASWHQHYMKHKP (SEQ ID NO: 12), SEFHRHGDKEHK (SEQ ID NO:13), CEFPRSWDMETN (SEQ ID NO: 14), SLTRHKPEPHRK (SEQ ID NO: 15),SLTWHKHELHRK (SEQ ID NO: 7), SPPWHKHELHRK (SEQ ID NO: 8), GGWHKHISRSDP(SEQ ID NO: 16), YHKHPHTYHNFK (SEQ ID NO: 17), HPKHPHTHTNDQ (SEQ ID NO:18), HMHMHQHVAQTQ (SEQ ID NO: 19), HMGMTKINYSAL (SEQ ID NO: 20),SNYSDVKRLPTV (SEQ ID NO: 21), and SVNWQKQTISNL (SEQ ID NO: 22). In yetanother embodiment, the protective monoclonal antibody is 1E4 and iscomprised of polypeptide sequences of SEQ ID NO: 1 and SEQ ID NO: 2, orpolypeptide sequences having at least 90% identity to the polypeptidesequences of SEQ ID NO: 1 and SEQ ID NO: 2. In another embodiment, theprotective monoclonal antibody 1E4 comprises nucleotide sequences of SEQID NO: 3 and SEQ ID NO: 4, or nucleotide sequences having at least 90%identity to the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4,which encode the polypeptide sequences of SEQ ID NO: 1 and SEQ ID NO: 2,respectively.

In another embodiment, there is a composition for protection against C.burnetti infection in a subject comprising a peptide with a sequence ofSLTWHKHELHRK (SEQ ID NO: 7) or SPPWHKHELHRK (SEQ ID NO: 8), or a peptidesequence having at least 90% identity to SLTWHKHELHRK (SEQ ID NO: 7) orSPPWHKHELHRK (SEQ ID NO: 8). In one embodiment, the compositioncomprises the peptide with the sequence SLTWHKHELHRK (SEQ ID NO: 7) thatis conjugated to Keyhole limpet hemocyanin (KLH). In another embodiment,the subject is one or more of mice, guinea pigs or humans. In a furtherembodiment, the peptide comprising the peptide sequence of SLTWHKHELHRK(SEQ ID NO: 7) binds to 1E4 antibody. In one embodiment, the peptidecomprising the peptide sequence of SLTWHKHELHRK (SEQ ID NO: 7)conjugated to KLH binds to 1E4 antibody. In another embodiment, thepeptide contains a W/HXH motif, where X represents any amino acid.

Although the peptide mimics have been considered as potential surrogateantigens of carbohydrates for vaccine development against severalmicroorganisms, only few studies have achieved protective efficacytesting of mimetic peptides. Previous studies suggested that the mostwidely used parameters for selecting peptide mimics, such ashigh-affinity binding to a relevant anti-carbohydrate antibody,competition with native carbohydrate antigen for antibody binding, andinduction of robust anti-peptide response, maybe insufficient and notpredictive of whether a mimetic peptide is capable of eliciting anprotective carbohydrate cross-reactive immune response.

The mimotope vaccine strategies are based on the concept of reversevaccinology, in which protective Abs can be used as probes to identifymimetic peptides that can re-induce protective Ab responses in vivo. Theaforementioned results against C. burnetii demonstrate that theidentification of protective mAb 1E4 led to the identification ofprotective mimic peptide m1E41920, suggesting that using protective Absas probes is critical to identify protective mimetic peptides.

In addition, the sequencing analysis and computational docking resultsprovide theoretical evidence to support that m1E41920 cannot onlysimulate the sugar cyclic structures through the imidazole ring ofhistidine in W/HXH motif, but also can simulate the mAb/LPS recognitionthrough binding of the same HCDR3 residues. These characteristics maycontribute more to the successful identification of the protective mimicpeptide in this location. The mimetic peptide m1E41920 fits well intothe binding site of the parent mAb 1E4 VH chain. The directly contactedm1E41920 and 1E4 VH residues were identified and there was no directcontacted residue in the 1E4 VL chain. The model docking structure ofthe 1E4-m1E41920 complex shows that the mimetic peptide m1E41920 fitsinto the H chain groove with the middle four-residue domain (WHKH) (SEQID NO: 28) in a helix turn, whereas it directly contacts HCDR1, HCDR2,and HCDR3 in an extended conformation through the N-terminal domainSLTWH (SEQ ID NO: 29) and C-terminal domain -L-R-, respectively. Thissuggests that the m1E41920 central domain is necessary to maintain theconformation, and the two terminal domains are involved in binding toHCDRs.

The automatic modeling of 1E4 variable domains was established bycanonical structure method from RosettaAntibody. The structure ofmimetic peptide m1E41920 was modeled using SAM-T08. The two predictedBrookhaven Protein Data Bank files were loaded onto the RosettaDock webserver to obtain the 1E4-m1E41920 complex model. Structure analysis andgraphical renderings of the top one docking prediction were performed byusing PyMOL.

In one embodiment, the protective epitopes on PI-LPS were identified toprove the concept that peptide mimics of PI-LPS can confer protectiveimmunity against C. burnetii infection; a novel protective mAb thatrecognizes a PI-specific epitope on PI-LPS was developed and identifieda protective peptide mimic of PI-LPS by screening a phage displaylibrary with the protective mAb. To our knowledge, this study providesthe first evidence to demonstrate that there is a protective epitope onPI-LPS and prove the feasibility of development of a peptide mimicvaccine against Q fever.

Several PI-LPS—specific mAbs have been developed for analysis ofantigenicity of immunogenic components of C. burnetii LPS and detectionof the virulent form of C. burnetii. Hotta et al. reported generation of19 PI-LPS—specific mAbs and demonstrated that these mAbs were able to beseparated into three groups based on their reactivity withO-polysaccharide chains (27 kDa of PI-LPS), O-polysaccharide chains(15-27 kDa of PI-LPS), and outer-core oligosaccharides (14 kDa ofPI-LPS). Palkovicova et al. developed a virenose-targeted mAb (IgG2bsubclass) that recognized O-polysaccharide chains and suggested thatthis virenose-unique mAb may be a useful biomarker for detection ofvirulent C. burnetii. However, it still remains unknown whetherPI-LPS—recognized mAbs are protective. The aforementioned methods may beemployed to identify and generate peptide-based vaccines againstSalmonella enteritidis, Mycobacterium tuberculosis, Francisellatularensis, Chlamydia trachomatis and Brucella abortus.

m1E41920 Fits well into the 1E4 Groove in the Docking Model

As shown in FIG. 13, the mimetic peptide m1E41920 fits well into thebinding site of the parent mAb 1E4 V_(H) chain. The directly contactedm1E41920 and 1E4 VH residues were identified and displayed as red andblue spacefill graphs, respectively (FIG. 13(A)), although there was nodirect contacted residue in the 1E4 V_(L) chain (data not shown). Themodel docking structure of the 1E4-mE41920 complex was displayed as acartoon graph in FIG. 13(B). The mimetic peptide m1E41920 fits into theH chain groove with the middle four-residue domain (WHKH) (SEQ ID NO:28) in a helix turn, whereas it directly contacts HCDR1, HCDR2, andHCDR3 in an extended conformation through the N-terminal domain SLTWH(SEQ ID NO: 29) and C-terminal domain L-R-, respectively. This suggeststhat the m1E41920 central domain is necessary to maintain theconformation, and the two terminal domains are involved in binding toHCDRs. It may also help to explain why m1E41920 is a stronger inhibitor,as only m1E41920 contains the complete motif SLTWH (SEQ ID NO: 29),which may be very important for the 1E4 binding. In addition, although1E4 HCDR3 has 15 residues (FIG. 13(B)), only the four HCDR3 centralresidues (VM-DY), which are de novo generated by D-J conjugation and Ninsertion, directly contact five N-terminal residues (SLTWH) (SEQ ID NO:29) of m1E41920 (FIG. 13(B)). This suggests that m1E41920 can bind tothe LPS binding site of 1E4. These data provide theoretical evidences tosupport the m1E41920 structural mimicry of PI-LPS based on its abilityto bind to the same HCDR3 residues of 1E4. For FIG. 13(A), the imageswere generated using Pymol, and the 1E4-m1E41920 interaction model wasshown as a spacefill graph. The directly contacted m1E41920 and 1E4V_(H) residues in direct contact were identified and displayed as redand blue spacefill graph, respectively. The contacted residues of the1E4 V_(H) paratopes in complex with m1E41920 are shown in blue, and theremainders are shown in green. The peptide mimic m1E41920 fits well intothe binding site of the parent mAb 1E4 V_(H) chain. For FIG. 13(B), themodel docking structure of the 1E4-m1E41920 complex was displayed as acartoon graph. The contacted residues and amino acid abbreviations areshown in the corresponding color described in (A). The peptide mimicm1E41920 fits into the H chain groove with the middle four residuedomain (WHKH) (SEQ ID NO: 28) in a helix turn, whereas it directlycontacts HCDR1, HCDR2, and HCDR3 in an extended conformation through theN-terminal domain SLTWH (SEQ ID NO: 29) and C-terminal domain L-R-,respectively.

Characterization of Fab1E4, muscFv1E4 and huscFv1E4

Expression and purification of recombinant muscFv1E4 and huscFv1E4:Escherichia coli strain BL21 was transformed with pETmuscFv1E4 orpEThuscFv1E4 and then incubated at 37° C. in 2×YT broth with ampicillin.When the optical density reached 0.6 at a wavelength of 600 nm, IPTG(isopropyl-1-thio-β-D-galactopyranoside) was added to the culture at afinal concentration of 1 mM and the cells were further grown overnightat 25° C. Pelleted cells were suspended in 10 ml of ice-cold periplasmicextraction buffer [30 mM Tris—HCl (pH 8.0), 20% (w/v) sucrose, 1 mMEDTA], 200 units of lysozyme (Epicentre, Madison, Wis.) were added andincubated at room temperature (RT) for 10 min. Lysozyme-treated bacteriawere pelleted by centrifugation and the supernatant was dialyzedovernight against PBS at 4° C. Recombinant muscFv1E4 and huscFv1E4 werepurified under native conditions using Ni-NTA HisBind Resins (Novagen)according to the manufacturer's instructions.

Purified 1E4, Fab1E4, muscFv1E4 and huscFv1E4 were resuspended inreduced loading buffer and separated by 12% SDS-PAGE gels using aMini-PROTEANII apparatus (Bio-Rad Laboratories). The purity of purified1E4, Fab1E4, muscFv1E4 and huscFv1E4 was analyzed by Coomassieblue-staining of SDS-PAGE gel. For Western blotting, samples wereseparated by SDS-PAGE and transferred electrophoretically ontonitrocellulose membranes in Tris-glycine buffer. The membranes wereblocked for 1 h at room temperature (RT) in PBS with 0.05% TWEEN 20(nonionic detergent) (PBST) and 10% nonfat dry milk and then incubatedwith 1:2000 diluted mouse anti-6×His epitope tag mAb (Thermo FisherScientific) at 4° C. overnight. After washing five times (5 min eachwash) with PBST buffer, the membranes were incubated with horseradishperoxidase (HRP)-conjugated goat anti-mouse IgG (1:10,000 dilution)(SouthernBiotech, Birmingham, Ala.) for 1 h at RT. The reactions weredetected by using an ECL Western blot detection kit (Thermo FisherScientific).

In order to examine the ability of Fab1E4, muscFv1E4 and huscFv1E4 toinhibit C. burnetii infection, the purity and specificity of purifiedFab1E4, muscFv1E4 and huscFv1E4 were analyzed by SDS-PAGE and Westernblotting. A shown in FIG. 14(A), two bands of 50 kDa heavy chains and 25kDa light chains were observed in 1E4 preparation (lane 1). Two similar˜25 kDa bands showing dissociated light (VL-Cκ) and heavy changes(VH-CH1) were detected in purified Fab1E4 fragments (lane 2). Asexpected, a 30 kDa band was observed in the purified recombinantmuscFv1E4 (lane 3) and huscFv1E4 (lane 4). FIG. 14(B) shows that both 50kDa heavy chains and 25 kDa light chains of 1E4 (lane 1) and both theVL-Cκ and VH-CH1 of Fab1E4 (lane 2) reacted with goat anti-mouse IgG inWestern blotting as predicted. Western blotting analysis also indicatedthat purified muscFv1E4 and huscFv1E4 reacted with anti-6×His mAb (FIG.14(B), lanes 3 & 4). In addition, the nucleotide sequencing results anddeduced amino acid sequences demonstrated that the assembled muscFv1E4gene was correctly constructed and that the variable regions of heavyand light changes were productively rearranged with the linker sequence(data not shown). These results confirmed the purity and specificity ofpurified Fab1E4, muscFv1E4 and huscFv1E4.

Next, the m1E41920 peptide was used to evaluate the binding ability ofFab1E4, muscFv1E4 and huscFv1E4 by indirect ELISA. As shown in FIG.14(C), 1E4, Fab1E4, muschFv1E4 and huscFv1E4 were able to bind tom1E41920 in a dose dependent manner but the binding capacity differedamong 1E4, Fab1E4, muscFv1E4 and huscFv1E4. The nonlinear regressionderived by GraphPad Prism analysis showed 1E4 had the lowest halfmaximal effective concentration (EC50, the concentration needed toachieve 50% maximal binding) of 6.82 nM to m1E41920, while the EC50swere 26.85 nM, 73.10 nM and 62.29 nM for Fab1E4, muscFv1E4 andhuscFv1E4, respectively. These results suggest that the bindingcapability to m1E41920 is similar between muschFv1E4 and huscFv1E4 butis lower than Fab1E4. In addition, we also examined whether Fab1E4,muscFv1E4 and huscFv1E4 can specifically bind to C. burnetii nativeantigen (PI whole cell antigen) by indirect ELISA. As shown in FIG.14(D), Fab1E4, muscFv1E4 and huscFv1E4 were able to specifically bind toboth PI whole cell antigen and m1E41920, however, their bindingcapability was lower than 1E4. These results suggest that Fab1E4,muscFv1E4 and huscFv1E4 retain a comparable binding activity to mimeticpeptide and native antigen as 1E4.

Indirect ELISA was performed as follows: Briefly, 5 μg/ml PI whole cellantigen or 50 μg/ml synthetic mimetic peptide in 100 μl 0.05 Mcarbonate/bicarbonate coating buffer (pH 9.6) was added to each well ofa 96-well microtiter plate and coated at 4° C. for 48 h. Plates werethen blocked with 1% BSA in PBST buffer (0.05% TWEEN 20 (nonionicdetergent) in PBS) for 1 hour at 37° C. and incubated with 100 ul ofdiffering concentrations of 1E4, Fab1E4, muscFv1E4 or huscFv1E4 at 4° C.overnight. After washing four times (5 min each wash) with PBST buffer,100 μl of anti-6×His mAb (1:2000) was added to each well and incubatedat 37° C. for 2 h. Following washing five times with PBST buffer, theplates were incubated with 100 ul of HRP-conjugated goat anti-mouse IgG(1:2000) at 37° C. for another 2 h. After washing five times with PBSTbuffer, the Sigma Fast O-Phenylenediamine Dihydrochloride Tablet Sets(Sigma-Aldrich) were used as substrates and the OD was measured at 490nm by the SPECTRA MAX M2 system (Molecular Devices Corporation,Sunnyvale, Calif.).

To determine whether Fab1E4, muscFv1E4 and huscFv1E4 retain the abilityof 1E4 to inhibit C. burnetii infection in vivo, we examined iftreatment of virulent C. burnetii with Fab1E4, muscFv1E4 or huscFv1E4would inhibit C. burnetii infection in mice. Before infection of micewith Ab treated C. burnetii, IFA was used to confirm whether 1E4,Fab1E4, muschFv1E4 and huscFv1E4 can specifically bind to live virulentC. burnetii. As shown in FIG. 15(A), the C. burnetii immune complexformation was detected in 1E4, Fab1E4, muscFv1E4 or huscFv1E4 treated C.burnetii but was not detected in IgG2a isotype control of PBS treated C.burnetii (data not shown). These results suggest that 1E4, Fab1E4,muscFv1E4 and huschFv1E4 were able to specifically bind to live C.burnetii. As shown in FIG. 15(B), compared to mice infected with PBS orIgG2a isotype control treated C. burnetii, splenomegaly wassignificantly reduced in mice infected with 1E4, Fab1E4, muscFv1E4 orhuscFv1E4 treated C. burnetii (p<0.001). In addition, splenomegaly inmice infected with huscFv1E4 treated C. burnetii was similar to miceinfected with Fab1E4 or muscFv1E4 treated C. burnetii but it wassignificantly greater than in mice infected with 1E4 treated C.burnetii. In support of the splenomegaly results, compared to miceinfected with PBS or IgG2a isotype control treated C. burnetii,significantly lower C. burnetii genome copies were detected in spleensfrom mice infected with 1E4, Fab1E4, muscFv1E4 or huscFv1E4 treated C.burnetii. While the C. burnetii load in spleen was not significantlydifferent among mice infected with Fab1E4, muscFv1E4 or huscFv1E4treated C. burnetii, it was significantly higher than in mice infectedwith 1E4 treated C. burnetii (FIG. 15C). FIG. 13D showshistopathological differences in the spleens from different groups ofmice. Large numbers of moderate to large accumulations of macrophages(FIG. 15(D), encircled by a black border) were present in the red pulpof spleens form mice infected with C. burnetii treated by PBS, IgG2aisotype control, Fab1E4, or muscFv1E4. In contrast, only few small tomoderate accumulations of macrophages (FIG. 15(D), encircled by a blackborder) appeared in the red pulp of spleens from mice infected with C.burnetii treated by 1E4 (p<0.001) or huscFv1E4 (p<0.01). As shown inFIG. 15(E), compared to mice infected with PBS or IgG2a isotype controltreated C. burnetii, the inflammation score in the spleen wassignificantly lower in mice infected with 1E4 or huscFv1E4 treated C.burnetii, however, it was similar in mice infected with Fab1E4, ormuscFv1E4 treated C. burnetii. These results indicate that althoughFab1E4, muscFv1E4 and huscFv1E4 were able to inhibit C. burnetiiinfection in vivo, their ability to inhibit C. burnetii infection waslower than 1E4, suggesting both the variable region and Fc fragment maybe required for 1E4 mediated protection in vivo. For FIGS. 15(B), 15(C)and 15(E), **p<0.01 and ***p<0.001.

Immunofluorescence assay (IFA): Infected cells were fixed with 2%paraformaldehyde for 15 min and permeabilized with cold methanol for 10min. Rabbit anti-PI polyclonal antibodies (1:500) were used to stainintracellular C. burnetii, followed by incubation with 10 μg/mlFITC-labeled goat anti-rabbit IgG (1:1,000) (Southern Biotechnology,Birmingham, Ala.). Host nuclei were stained by DAPI in mounting medium(1:500) (Invitrogen) and slides were examined by using fluorescencemicroscopy. In addition, IFA was also used to detect the C. burnetiiimmune complex formation. Briefly, live virulent C. burnetii organisms(1×10⁷) were incubated in 500 μl PBS with 1% BSA containing differentconcentrations of IgG2a isotype control, 1E4, Fab 1E4, muscFv1E4 orhuscFv1E4 at 4° C. overnight. The mixture was spun at 15,000 rpm/min ina microfuge for 30 min, the pellets were spread on a cover slide andthen fixed with 2% paraformaldehyde for 15 min. For the IFA, the pelletsfrom the mixture of C. burnetii with IgG2a, 1E4 or Fab1E4 were directlystained with FITC labeled goat anti-mouse IgG, while the pellets fromthe mixture of C. burnetii with muscFv1E4 or huscFv1E4 were incubatedwith mouse anti-6×His mAb first and then stained with FITC labeled goatanti-mouse IgG. The binding of IgG2a isotype control, 1E4, Fab1E4,muscFv1E4 or huscFv1E4 with live virulent C. burnetii was examined byusing fluorescence microscopy.

Quantitative PCR assay: High Pure PCR Template Preparation Kit (RocheMolecular Biochemicals, Indianapolis, Ind.) with modifications was usedfor extraction of DNA templates from C. burnetii infected mouse tissuesand cells. Real time-PCR was performed using Applied Biosystems7300/7500 Real Time PCR System. The recombinant plasmid DNA (com1 geneligated into pET23a vector) was used as a standard DNA to quantify com1gene copy numbers in spleen samples.

To determine whether Fab 1E4, muscFv1E4 and huscFv1E4 retain the abilityof 1E4 to inhibit C. burnetii infection in vivo, we examined iftreatment of virulent NMI with Fab 1E4, muscFv1E4 or huscFv1E4 wouldinhibit C. burnetii infection in BALB/c mice. In order to compare theinhibition ability of Fab1E4, muscFv1E4 and huscFv1E4 with 1E4 in vivo,it is important to make sure that the same numbers of C. burnetiitreated with different Ab will be injected into mice. Since aerosolinfection cannot guarantee that mice will receive the same numbers of Abtreated C. burnetii between different experimental groups, thisexperiment was performed by i.p. injection. Since one molecule of 150kDa 1E4 carries two antigen binding sites, and one molecule of either 50kDa Fab1E4, 25 kDa muscFv1E4 or 25 kDa huscFv1E4 carries only oneantigen binding site, inhibition of C. burnetii with 1E4, Fab 1E4,muscFv1E4 or huscFv1E4 was performed by incubating 1×10⁷ virulent C.burnetii NMI with 300 μg of 1E4, 200 μg of Fab 1E4, 100 μg of muscFv1E4or 100 μg of huscFv1E4 (which contained the same antigen binding sites)at 4° C. overnight. Six week-old BALB/c mice were infected by i.p.injection with 1×10⁷ of 1E4, Fab1E4, muscFv1E4 or huscFv1E4 treated C.burnetii. The respective abilities of 1E4, Fab1E4, muscFv1E4 orhuscFv1E4 to inhibit C. burnetii infection in BALB/c mice were evaluatedby comparing splenomegaly, bacterial burden and histopathologicalchanges in the spleen at 14 days post infection with PBS and mouse IgG2aisotype control.

To determine whether 1E4, Fab1E4, muscFv1E4 and huscFv1E4 can block C.burnetii infection in vitro, we examined if treatment of C. burnetiiwith 1E4, Fab1E4, muscFv1E4 or huscFv1E4 could inhibit C. burnetiiinfection in Bone Marrow-Derived Macrophages (BMDM). Bone marrowprecursor cells were counted and placed into a culture flask in RPMI1640 medium containing 10% fetal bovine serum and 30% of L929 cellconditioned medium (LCCM) at a concentration of 2×10⁶ cells/ml andincubated at 37° C. for 7 days. Human monocyte-like (THP-1) cells(TIB-202; ATCC) were maintained in RPMI 1640 medium (Invitrogen,Carlsbad, Calif.) supplemented with 10% fetal calf serum (HyClone,Logan, Utah) at 37° C. in 5% CO₂. THP-1 cells were differentiated intoadherent, macrophage-like cells by treating freshly plated cells withPMA (200 nM; EMD Biosciences, San Diego, Calif.) for 3 days. For invitro assay, BMDM or THP-1 derived human macrophages were removed fromthe substrate by HyQTase (HyClone) digestion and added to glasscoverslips inserted in 24-well tissue culture dishes at a density of2×10⁵ per well. Compared to macrophages infected with PBS or IgG2aisotype control treated C. burnetii, fewer C. burnetii infectedmacrophages were observed in macrophages infected with Fab1E4, muscFv1E4or huscFv1E4 treated C. burnetii while more C. burnetii infectedmacrophages were observed in macrophages infected with 1E4 treated C.burnetii (FIG. 16(A)). As shown in FIG. 16(B), compared to macrophagesinfected with PBS or IgG2a isotype control treated C. burnetii, theinfection rate was significantly (P<0.001) decreased in macrophagesinfected with Fab1E4, muscFv1E4 or huscFv1E4 treated C. burnetii but itwas significantly (P<0.001) increased in macrophages infected with 1E4treated C. burnetii at 1 day post infection. In addition, C. burnetiigenome copies in C. burnetii infected macrophages were measured byreal-time-PCR at 1 day post infection. As shown in FIG. 16(C), comparedto macrophages infected with PBS or IgG2a isotype control treated C.burnetii, the C. burnetii genome copies were significantly (P<0.001)lower in macrophages infected with Fab1E4, muscFv1E4 or huscFv1E4treated C. burnetii but were significantly (P<0.01) higher inmacrophages infected with 1E4 treated C. burnetii. Interestingly, therewas no significant difference in C. burnetii infection rate and genomecopies among macrophages infected with Fab 1E4, muscFv1E4 and huscFv1E4treated C. burnetii. These results indicate that Fab1E4, muscFv1E4 andhuscFv1E4 can block C. burnetii infection in vitro though their parentmAb 1E4 did not. The observation that C. burnetii infection rate andgenome copies in macrophages infected with 1E4 treated C. burnetii weresignificantly higher than macrophages infected with PBS or IgG2a isotypecontrol treated C. burnetii suggests that macrophages were able touptake Ab-C. burnetii immune complex via Fc receptor-mediatedphagocytosis.

C. burnetii inhibition assay in vitro: BMDM were used to examine iftreatment of C. burnetii with 1E4, Fab1E4, muscFv1E4 or huscFv1E4 caninhibit C. burnetii infection in vitro. C. burnetii NMI was treated with1E4, Fab 1E4, muscFv1E4 or huscFv1E4 in the same manner as describedabove and inoculated with macrophages at MOI of 100 and incubated at 37°C. for 2 h. After three times gentle washing with warm media to removefree bacteria, infected macrophages were cultured at 37° C. for 24 h. C.burnetii infection rate was determined at 24 h post infection byindirect IFA and the C. burnetii genomic copy numbers in macrophageswere measured by Quantitative real time PCR assay. In addition, THP-1derived human macrophages were used to determine whether huscFv1E4 canneutralize C. burnetii to block C. burnetii infection in human cells. C.burnetii treatment with huscFv1E4 and inoculation of human macrophageswere performed in a similar manner as described in mouse macrophages.

To determine whether huscFv1E4 can neutralize C. burnetii to block C.burnetii infection in human cells, THP-1 cell derived human macrophageswere infected with PBS, IgG2a isotype control or huscFv1E4 treated C.burnetii. As shown in FIG. 17(A), compared to human macrophages infectedwith PBS or IgG2a isotype control treated C. burnetii, fewer C. burnetiiinfected macrophages were observed in macrophages infected withhuscFv1E4 treated C. burnetii at 1 day post infection. In addition, theC. burnetii infection rate and genome copies in human macrophagesinfected with huscFv1E4 treated C. burnetii were significantly (P<0.001)lower than macrophages infected with PBS or IgG2a isotype controltreated C. burnetii (FIGS. 17(B) & (C)). These results demonstrate thathuscFv1E4 was able to neutralize C. burnetii to block C. burnetiiinfection in human cells and suggest that huscFv1E4 can be used toprevent human Q fever.

To determine whether 1E4, Fab1E4, muscFv1E4 and huscFv1E4 can directlykill C. burnetii, viable and nonviable bacteria in 1E4, Fab1E4,muscFv1E4 or huscFv1E4 treated C. burnetii were stained with BacLightkit and analyzed by fluorescence microscopy. In this assay, bacteriawere stained with a green fluorescent dye (SYTO9) and a red fluorescentdye (PI). Since SYTO9 can penetrate all bacterial cells but PI onlypenetrates membrane damaged bacteria, viable bacteria will be stained bySYTO9 as green color while dead bacteria will be stained by PI as redcolor. More viable bacteria (green) were observed in 1E4 treated C.burnetii but more dead bacteria were found in EDTA treated C. burnetii(data not shown). Compared to PBS or IgG2a isotype control treated C.burnetii, a similar number of dead bacteria was found in 1E4, Fab 1E4,muscFv1E4 or huscFv1E4 treated C. burnetii but significantly highernumbers of dead bacteria (p<0.001) were observed in EDTA treated C.burnetii. These results suggest that neither 1E4 nor Fab1E4, muscFv1E4and huscFv1E4 can mediate direct killing of virulent C. burnetii.

Fluorescence microscopic assay of viable and nonviable C. burnetiicells: Approximately 1×10⁷ virulent C. burnetii PI organisms wereincubated with 1E4, Fab1E4, muscFv1E4 or huscFv1E4 in the same manner asdescribed above or IgG2a isotype control in 500 ul PBS at 4° C.overnight. In addition, C. burnetii treated with 10 mM EDTA at 4° C. for1 h was used as positive controls. C. burnetii was pelleted at 15,000rpm/min in a microfuge for 30 min, washed and resuspended in sterile0.85% NaCl. The BacLight stock solution A (SYTO9) and B (PI)(Invitrogen) were added and incubated in the dark at room temperaturefor 15 min according to the manufacturer's instructions. The stainedsolution was mounted in BacLight mounting oil (Invitrogen) on a clearglass slide. A total of 100 PI organisms were counted per slide at1,000× using a fluorescence microscope and the duplicate numbers of deadbacteria (stained red) were recorded.

Histopathology: Lungs and spleens were collected from mice at 14 dayspost challenge with C. burnetii, fixed in 10% formalin—PBS at least for48 h, prepared as 5-μm paraffin-embedded sections by standard methods,and then sliced. Slides were stained with hematoxylin and eosin andexamined in a blinded fashion for evaluation of histopathology.

Statistical analysis: Statistical comparisons were performed with Prism5.0 (GraphPad Software Inc., San Diego, Calif.). Results expressed asmeans±standard deviations were compared with the two-sample Student'st-test or one-way ANOVA and the post test. Differences were consideredsignificant at p<0.05.

In one embodiment, there is a composition for protection against C.burnetti infection in a subject comprising monoclonal antibodies 1E4,muscFv1E4 or huscFv1E4. In another embodiment, the monoclonal antibodiesmuscFv1E4 and huscFv1E4 comprise a polypeptide sequence of SEQ ID NO: 5and SEQ ID NO: 6, respectively, or a polypeptide sequence having atleast 90% identity to SEQ ID NO: 5 and SEQ ID NO: 6. In anotherembodiment, the monoclonal antibodies muscFv1E4 and huscFv1E4 arecapable of binding to m1E41920. In one embodiment, the monoclonalantibodies muscFv1E4 and huscFv1E4 are capable of binding to live C.burnetii. In one embodiment, the monoclonal antibodies muscFv1E4 andhuscFv1E4 are capable of inhibiting C. burnetii infection of cellculture in vitro. In another embodiment, the monoclonal antibodieshuscFv1E4 can inhibit C. burnetii infection of human macrophages invitro. In one embodiment, the monoclonal antibodies huscFv1E4 caninhibit C. burnetii infection of human macrophages in vivo.

Despite C. burnetii being an obligate intracellular pathogen, previousstudies have shown that passive transfer of Abs was able to confersignificant protection against C. burnetii intraperitoneal infection inmice, demonstrating the possibility of development of Ab-basedimmunotherapeutic strategies to prevent human Q fever. Since passiveadministration of Abs can provide immediate immunity against biologicalagents and there is no licensed vaccine available for protection of Qfever in the US, development of Ab-based immunotherapeutic strategies toprevent human Q fever has a high potential impact for public health andbiodefense against C. burnetii natural infection and the use of C.burnetii for biological warfare. Our recent study demonstrated thattreatment of C. burnetii with the PI-LPS specific mAb 1E4 was able toinhibit C. burnetii infection in mice in a dose-dependent manner,suggesting that 1E4 is a protective mAb. However, it remains unknownwhether passive administration of 1E4 will provide significantprotection against C. burnetii natural infection. To prove thefeasibility of using 1E4 to prevent C. burnetii natural infection, weexamined if passive transfer of 1E4 would provide significant protectionagainst C. burnetii aerosol challenge in SCID mice. The result indicatedthat passive transfer of 1E4 was able to confer significant protectionagainst aerosolized C. burnetii in SCID mice. Since SCID mice lackfunctional B and T cells which may mimic chronic Q fever patients withimmunocompromised conditions, this finding suggests that 1E4 may beuseful to protect individuals with immunocompromised conditions againstC. burnetii natural infection. To our knowledge, this is the firstevidence demonstrating that passive transfer of Abs can provideprotection against C. burnetii natural infection. Since C. burnetiiinfection in individuals with immunocompromised conditions can developinto more severe and fatal chronic diseases and there is no licensedvaccine available for protection of Q fever in the US, developing ahumanized antibody to use as a prophylactic agent is important forprotection of individuals at high risk of exposure from developingchronic Q fever.

One early study demonstrated that purified human anti-PI IgM was able tosuppress C. burnetii replication in the mouse spleen when mixed with asuspension of organisms prior to inoculation of mice. In addition, ourrecent studies have shown that treatment of C. burnetii with 1E4 orpurified IgM or IgG from formalin-inactivated PI vaccine immunized mousesera was able to inhibit C. burnetii infection in BALB/c mice. This datasuggests that anti-PI Abs may be able to inhibit C. burnetii infectionvia their ability to neutralize or kill the organisms. However, therehas been no direct evidence to support this hypothesis. To address thisquestion, we isolated the Fab fragment of 1E4 (Fab1E4) and examined ifFab 1E4 binding with C. burnetii would block C. burnetii infection inboth in vitro and in vivo systems. The results indicated that Fab1E4retained a binding capability comparable to 1E4 and was able toneutralize virulent C. burnetii resulting in inhibiting C. burnetiiinfection in both in vitro and in vivo systems. These observationsprovided clear evidence to support that anti-PI Abs are able to inhibitC. burnetii infection via their ability to neutralize the organisms. Ithas been shown that LPS was involved in the virulent C. burnetii NMIuptake by either mouse or human macrophages through binding to integrinand/or TLR4 receptors (26). Since C. burnetii is smaller (0.2-2 μm) thantypical gram-negative bacteria (1-10 μm) and LPS covers most of thesurface of NMI, it is possible that NMI cells might be easily occupiedand coated by LPS specific antibodies. As 1E4 specifically recognizesPI-LPS, our results suggest that Fab1E4-mediated inhibition of C.burnetii infection might be due to its ability to bind with PI-LPSthereby blocking C. burnetii infection in susceptible host cells. Inaddition, we also investigated whether Ab binding with virulent C.burnetii can mediate direct killing of bacteria. The results suggestthat Ab was unable to mediate direct killing of virulent C. burnetii.These findings provide direct evidence to demonstrate that anti-PI Absmediated inhibition of C. burnetii infection is dependent on theirability to neutralize the organisms to block the infection but notdependent on their ability to directly kill bacteria.

The classic concept in our understanding of Ab structure and function isthat the variable region determines the antigen binding, whereas the Fcsegment determines the isotype, pharmacological characteristics andinteraction with Fc receptors. The best documented direct effect of thevariable region is virus neutralization, which is defined as theabrogation of virus infectivity by inhibiting virus attachment and earlyentry into susceptible host cells. In contrast, only a few studies havereported that the ability of Abs to block receptors is required for theuptake of intracellular bacterial pathogens and thereby inhibitingbacterial infection. In the present study, to determine the role of thevariable region and Fc segment in 1E4 mediated passive protection, weexamined if the variable region of 1E4 (Fab1E4) retains the ability of1E4 to inhibit C. burnetii infection in both in vitro and in vivosystems. The in vivo C. burnetii inhibition experiment demonstrated thatFab1E4 was able to inhibit C. burnetii infection in mice but its abilityto inhibit C. burnetii infection was lower than 1E4, suggesting thatboth the variable region and Fc segment may contribute to 1E4 mediatedpassive protection. The in vitro C. burnetii inhibition experimentindicated that compared to macrophages infected with PBS or IgG2aisotype control treated C. burnetii, the infection rate and genomecopies were significantly decreased in macrophages infected with Fab1E4treated C. burnetii but they were significantly increased in macrophagesinfected with 1E4 treated C. burnetii, suggesting that Fab1E4 inhibitedC. burnetii infection in vitro but 1E4 did not. Thus, the resultsregarding Fab1E4 mediated protection from the in vitro experimentcorrelated to the results from the in vivo experiment and support thatFab 1E4 was able to significantly inhibit C. burnetii infection.However, the results regarding 1E4 mediated protection from the in vitroexperiment do not support that 1E4 had a higher ability than Fab 1E4 toinhibit C. burnetii infection in mice. The observation that theinfection rate and genome copies were significantly increased inmacrophages infected with 1E4 treated C. burnetii correlated withseveral previous in vitro studies, suggesting that anti-C. burnetiispecific Abs can increase the ability of phagocytes to uptakeAb-opsonsized C. burnetii. This result can be explained by thepossibility that 1E4-opsonsized C. burnetii may enhance phagocyticactivity of macrophages via Fc receptor-mediated effects resulting inthe increased infection rate and genome copies in macrophages. Inaddition, the conflicting results regarding 1E4 mediated protectionbetween in vivo and in vitro model systems may be due to the in vitro C.burnetii inhibition assay's inability to mimic the in vivo situation andit is difficult to use a cell culture system to rule out what happenedafter macrophages uptake Ab-opsonsized C. burnetii in mice. Futurestudies to determine whether 1E4 and Fab 1E4 treated C. burnetii candifferentially stimulate immune response in mice and/or activatemacrophages would be helpful to further understand the mechanisms of1E4-mediated protective immunity against C. burnetii infection.

As we know, passive administration of mouse mAbs to humans is not safebecause it can induce human anti-mouse Ab responses. Thus, although ourresults demonstrated that passive transfer of 1E4 can providesignificant protection against C. burnetii natural infection in mice,1E4 cannot be directly used to prevent Q fever in humans or treat Qfever patients. To further demonstrate the feasibility of usinghumanized 1E4 to prevent human Q fever, we generated an recombinanthumanized Fab fragment of 1E4 (huscFv1E4) and examined if huscFv1E4retains the ability of 1E4 to inhibit C. burnetii infection in mice andmouse macrophages. The results indicated that treatment of C. burnetiiwith huscFv1E4 significantly inhibited the C. burnetii infection in bothin vitro and in vivo systems and there was no significant differencebetween huscFv1E4 and Fab 1E4 in their ability to inhibit C. burnetiiinfection. In addition, our results also demonstrated that huscFv1E4 wasable to neutralize C. burnetii to block C. burnetii infection in humanmacrophages. These results suggest that huscFv1E4 may be useful forpreventing human Q fever. However, the observation that huscFv1E4 hadlower inhibition capability than 1E4 in mice suggests that Fc segmentmay be required for 1E4-mediated complete passive protection. These datademonstrate that CDR grafting humanization retained comparable levels ofprotective ability of mouse mAb in both in vitro and in vivo systems andprove the feasibility of the generation of a fully humanized mAb 1E4 forpreventing human Q fever.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the practice of the presentmethod and in construction and use of the present technology departingfrom the scope or spirit of the invention. Other embodiments of theinvention will be apparent to those skilled in the art fromconsideration of the specification and practice of the invention. It isintended that the specification and examples be considered as exemplaryonly.

What is claimed:
 1. An antibody or fragment thereof that binds to alipopolysaccharide-based epitope in gram negative bacteria, wherein theantibody or fragment thereof comprises the polypeptide sequence of SEQID NO: 5 or SEQ ID NO:6.
 2. The antibody or fragment thereof of claim 1,wherein the antibody or fragment thereof competes for binding to one ormore of the following peptide sequences: (SEQ ID NO: 9) 1.)SWFHPQRRHSHQ, (SEQ ID NO: 10) 2.) SWMPHPRWSPQH, (SEQ ID NO: 11) 3.)MHRAPSTHKLLP, (SEQ ID NO: 12) 4.) ASWHQHYMKHKP, (SEQ ID NO: 13) 5.)SEFHRHGDKEHK, (SEQ ID NO: 14) 6.) CEFPRSWDMETN, (SEQ ID NO: 15) 7.)SLTRHKPEPHRK, (SEQ ID NO: 16) 8.) GGWHKHISRSDP, (SEQ ID NO: 17) 9.)YHKHPHTYHNFK, (SEQ ID NO: 18) 10.) HPKHPHTHTNDQ, (SEQ ID NO: 19) 11.)HMHMHQHVAQTQ, (SEQ ID NO: 20) 12.) HMGMTKINYSAL, (SEQ ID NO: 21) 13.)SNYSDVKRLPTV, (SEQ ID NO: 22) 14.) SVNWQKQTISNL, (SEQ ID NO: 7) 15.)SLTWHKHELHRK, or (SEQ ID NO: 8) 16.) SPPWHKHELHRK.


3. The antibody or fragment thereof of claim 1, wherein the antibody orfragment thereof comprises the polypeptide sequence of SEQ ID NO:
 6. 4.The antibody or fragment thereof of claim 1, wherein the antibody orfragment thereof comprises the polypeptide sequence of SEQ ID NO: 5.